http://confocal-microscopy-list.275.s1.nabble.com/Measuring-PSFs-using-water-dipping-objective-tp7578494p7578499.html
I can think of two possible issues afecting the result, very thick layer of agar being cracked by the objective, very high power output from the laser setting free the particles, if you keep the power low and change the wavelength you can avoid that one. I hope this can help you. Regards Gabriel OH
> From:
[hidden email]
> Subject: Re: Measuring PSFs using water dipping objective
> To:
[hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hi Stephan,
>
> > I am looking for a good protocol to measure PSFs on a confocal microscope
> > using water dipping objectives (objectives used for physiology, i.e. no
> > cover glass!).
> > I tried using fluorescent beads embedded in 5 % low melting point agarose,
> > but it appears that the beads are moving, probably due to slight swelling of
> > the agarose which is immersed in the water.
>
> I did something like that recently, using 80 nm diameter nanogold
> particles embedded in low-melt agarose and imaged in reflected light
> mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
> a fluorescent bead is that the reflected signal is bright and of
> narrowly defined spectral range (we wanted to define chromatic
> aberration of our objectives, not get an experimental PSF for
> deconvolution). Particle movement didn't seem to be too much of an
> issue, perhaps because I could scan fast thanks to the high signal. Or
> possibly, the agarose mixture was a bit different to yours. Or, it might
> not have mattered for our question and I have forgotten. If I scrape my
> neurons a bit harder I recall that the near-IR laser made the particles
> wobble a bit, maybe because of local heating. Let me know if you want
> details of the protocol.
>
> Michael